Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

Mounting attention has been focused on defects of the autophagy-lysosomal pathway in sepsis, however, the precise mechanisms governing the autophagy-lysosomal process in sepsis are poorly known. We have previously reported that Erbin deficiency aggravated the inflammatory response and organ injuries caused by sepsis. In the present study, we found that Erbin knockout impaired the autophagy process in both muramyl dipeptide (MDP)-induced bone marrow-derived macrophages (BMDMs) and sepsis mouse liver and lung, as detected by the accumulation of LC3-II and SQSTM1/p62, and autophagosomes. Pretreatment with autophagy inhibitor chloroquine (CQ) further aggravated inflammatory response and organ injuries in vivo and in vitro sepsis model. We also observed that the impaired lysosomal function mediated autophagic blockade, as detected by the decreased expression of ATP6V, cathepsin B (CTSB) and LAMP2 protein. Immunoprecipitation revealed that the C-terminal of Erbin (aa 391–964) interacts with the N-terminal of transcription factor EB (TFEB) (aa 1–247), and affects the stability of TFEB-14-3-3 and TFEB-PPP3CB complexes and the phosphorylation status of TFEB, thereby promote the nucleus translocation of TFEB and the TFEB target genes transcription. Thus, our study suggested that Erbin alleviated sepsis-induced inflammatory responses and organ injuries by rescuing dysfunction of the autophagy-lysosomal pathway through TFEB-14-3-3 and TFEB-PPP3CB pathway.


Introduction
Autophagy is a dynamic lysosome-dependent conserved catabolic process responsible for processing damaged organelles, metabolizing macromolecules and intracellular microbes to maintain cellular homeostasis and playing an essential role in cancer, neurodegenerative, metabolic, and inflammatory disorders [1,2].Autophagy could coordinate macrophage polarization, inflammasome activation and release of inflammatory cytokines, and tissue injury during sepsis [3].Classical autophagy/macroautophagy initiates the generation of a double-membrane vesicle called the autophagosome.Then the outer membrane of the autophagosome fuses with the lysosomal membrane to form an autolysosome, in which the cytosolic materials are degraded by the cathepsins and hydrolytic enzymes [4].Any perturbed step might result in the dysfunction of autophagy and human diseases.
The current new view is that lysosomes are no longer just static organelles that process and recycle cellular waste but highly dynamic structures that mediate the adaptation of cell metabolism to the environment [5,6].Lysosomes are acidic organelles containing more than 50 different hydrolases, and proper function of these enzymes requires a low pH (4.5-5.0)within the lysosome, maintained by empty Vacuolar-type proton pumping ATPase (V-ATPase), providing a suitable condition for lysosomal degradation of its contents [7].In the course of autophagy, the process of degradation and recycling strictly depends on the lysosomal function, as the contents in the autophagosomes are eventually degraded by lysosomes via autophagosome-lysosome fusion [8][9][10].Xia et al. [11] found that the V-ATPase 6V0D2 gene defect impaired the acidic environment of macrophage lysosome, led to the accumulation of autophagy proteins LC3-II and p62 in macrophages and the blockage of autophagic flux, and aggravated the mitochondrial damage and inflammatory response.In addition, they found that V-ATPase 6V0D2 gene knockout also aggravated Dextran sulfate sodium (DSS) -induced colitis in mice and Salmonella typhi-induced death in animals.
Lysosomes can mediate signaling pathways and transcriptional programs that sense the metabolic state of cells by regulating lysosomal biogenesis and autophagy, most of which depend on transcription factor EB (TFEB) [8].TFEB is one of the MiT-TFE family members, which can combine a palindromic DNA sequence located in the proximal promoter of target genes and regulate the expression of lysosomal and autophagy-related genes [12].Normally, phosphorylated TFEB binds to the molecular chaperone YWHA/14-3-3 in the cytoplasm.Under stress conditions, such as starvation or infection, cytoplasmic TFEB is rapidly dephosphorylated.The dephosphorylated TFEB dissociates from molecular companion protein 14-3-3 and transfers from cytoplasmic into intracellular nuclei, activating lysosomal and autophagy-related gene expression.Meanwhile, the balance between phosphorylation and dephosphorylation of TFEB by PPP3CB determines cytoplasmic and nucleus distribution [13][14][15].
Erbin, a newly identified PDZ protein that acts as an adaptor for the ERBB2/HER2 in epithelia, contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ domain at its carboxy terminus [16].Erbin was discovered as a regulator of inflammation response due to its ability to bind specifically to intracellular receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2), which is one of the pattern recognition molecules (PRMs), detecting peptidoglycan (PGN) through the recognition of muramyl dipeptide (MDP) common to bacteria, and then up-regulation of proinflammatory cytokines [17,18].The present study showed that Erbin alleviated the inflammatory response and organ injuries caused by sepsis by promoting lysosomal biogenesis and autophagy, bridging the immune system and the core autophagosomal machinery.Mechanically, Erbin interacts with TFEB and affects the stability of TFEB-14-3-3 and TFEB-PPP3CB complexes, promoting the activation of TFEB and the nucleus translocation, thus regulating the lysosomal biogenesis.Our results revealed a novel mechanism of autophagy activation regulated by Erbin protein, leading to the suppression of inflammatory response and avoiding organ injuries of sepsis.

Materials
MDP was purchased from Sigma-Aldrich (A9519).Lipofectamine-3000 (L3000015) was purchased from Invitrogen Corporation.Rapamycin (RAPA) was purchased from Solarbio (R8140).Chloroquine (CQ) and M-CSF were purchased from MedChemExpress (HY-W031727).Lysotracker Red (C11046) and Hoechst 33342 (C1022) were purchased from Beyotime Biotechnology.Unless specified otherwise, all biochemical reagents were purchased from Sigma-Aldrich.Mammalian expression plasmids for HA-tagged Erbin, Myc-tagged TFEB, and its mutants were constructed by standard molecular biology techniques.To verify constructs and the specificity of antibodies, all constructs were transfected into 293T cells, and expression was analyzed using Western blot.All constructs were confirmed by DNA sequencing.

Cells culture and treatment
Bone marrow-derived macrophages (BMDMs) were collected from the femurs of Erbin fl/fl and Erbin fl/fl−Lyz2−Cre mice, and cells were simultaneously lysed in 1 × ACK buffer.Then, the BMDMs were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml of penicillin and 100 μg/ml of streptomycin, and M-CSF (20 ng/ml) in a humidified atmosphere containing 5% CO 2 at 37 °C.7 days later, the supernatant was removed, and the cells were harvested via trypsin digestion and cultured for further experiments.Cells were treated with MDP (10 μg/ ml) for 6 h with or without various agonists or inhibitors according to the experimental design.Cells were collected and homogenized immediately to extract RNA or protein or fixed neutral-buffed formalin for staining.Cells were stained with LysoTracker Red to detect the acidic microenvironment of the lysosome.Confocal images were obtained using an FV10i FLUOVIEW Confocal Microscope (Olympus, Tokyo, Japan).The human monocytic cell line THP-1 was a gift from Pro. Ying Zhu of Wuhan University.THP-1 cells were differentiated to macrophages with 60 nM TPA for 12-14 h, and cells were cultured for 24 h without TPA.THP-1 cells were cultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat-inactivated FBS (Sigma, USA).HeLa and HEK293T cells were from the American Type Culture Collection.The HIV molecular clones pNL-4-3∆Env or control lentiviral vector were transfected in 293T cells with vesicular stomatitis virus-G envelope.After 24 h of transfection, the supernatant was collected, filtered, and normalized for viral budding by ELISA (ZeptoMetrix).

Animals
Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.Erbin fl/ fl/Lyz2−Cre mice were obtained by crossing the Erbin flox mice with Lyz2-Cre mice.Erbin whole gene knockout mice in the C57BL/6 background were generated by Wuhan Xianran Biological Technology Co. LTD.Erbin −/− mice were bred with C57BL/6 mice to obtain Erbin ± heterozygotes.Erbin −/− mice and littermate control obtained from heterozygote crosses were used for all experiments.All mice were male, eight weeks old at the time of use.Mice were maintained in specific pathogenfree conditions under a 12-h/12-h light/dark cycle and allowed ad libitum access to water and a standard laboratory diet but deprived of food just before cecal ligation and puncture (CLP).Control mice were also deprived of food in the same manner.All procedures and treatments were conducted by the ethical regulations set by the Animal Experimentation Committee of Wuhan University (WQ20210298).

Cecal ligation and puncture
Mice were anesthetized with sevoflurane.A 1-to 2-cm ventral midline incision was performed, and the cecum was isolated.The feces at the upper end of the cecum were gently squeezed to fill the end of the cecum.Ligation was performed at the midpoint of the cecal valve and cecum with sterile No.4 silk thread, and a 21G sterile needle was used to puncture the cecum at the midpoint of the ligation site and the top of the cecum.Gently squeeze the cecum, extrude a little content to ensure the perforation is unobstructed, wipe the extruded content, then push the cecum back to the abdominal cavity, close the abdominal cavity, and layer by layer suture.Postoperative fluid resuscitation was performed with normal saline (37 °C, 5 ml/100 g) and buprenorphine (0.05 mg/kg) for analgesia.Sham-operated animals received the same surgical procedures without ligation and puncture.Animals had free access to food and water postoperatively.To inhibit autophagosome-lysosome formation during sepsis, chloroquine (CQ) (60 mg/kg) was pre-injected intraperitoneally.Mice were subjected to CLP and killed at predetermined times to examine time-dependent effects.The serum was collected for biochemical analysis.The liver and lungs were collected and frozen immediately in liquid nitrogen for western blot or fixed in neutral-buffered formalin for histochemical examination.

Bronchoalveolar lavage fluid (BALF) collection and inflammatory cell counting and protein concentration determination
Mice were euthanized at the experimental time, and the lungs were lavaged with 1 ml PBS through the tracheal cannula to obtain BALF.The BALF was centrifuged at 800 × for 15 min at 4 °C, and cell pellets were resuspended in PBS.The total number of inflammatory cells in the BALF was determined by counting the cells with a hemocytometer.The polymorphonuclear neutrophils (PMNs) cells were fixed and stained using Wright Stain solution.Then they were classified by a laboratory technologist blinded to the experimental design to determine the percentage of neutrophils.The total protein concentration in the supernatants was determined by the BCA method.

Western blot and immunoprecipitation assay
Immunoprecipitation was performed by lysing cells in IP buffer (50 mM Tris-HCI 8.0, 5 mM NaCl, 1 mM EDTA, 0.1% NP40).Cell lysates containing 1 mg protein of each treatment were incubated at 4 °C with primary antibody for 4 h before Dynabeads protein G (Thermo Scientific) was added to the samples.After further incubation for 4 h at 4 °C, beads were washed three times in ice-cold lysis buffer, and the immunoprecipitated complexes were eluted by boiling for 5 min in 1.5 × SDS buffer.Lastly, the eluted immunoprecipitated complexes were resolved for immunoblotting analysis.

Immunofluorescence
BMDMs were seeded at 1 × 10 5 cells on the coverslips and rested overnight for proper attachment.After treatment, cells were washed twice with sterile PBS, fixed with 4% ice-cold paraformaldehyde (PFA), then permeabilized with 0.25% Triton X-100 and blocked in 3% bovine serum albumin (BSA).Incubated with primary antibody overnight at 4 °C.The next day, coverslips were incubated with FICT-conjugated secondary antibody for 1 h at 37 °C.The coverslips were stained with DAPI and photographed under fluorescence microscopy.

Transmitted electron microscopy
BMDMs were fixed in a fixative buffer containing 2.5% buffered glutaraldehyde and 2% paraformaldehyde in 0.1 M of phosphate-buffered solution at 4 ℃ for 1 h, then fixed with 1% osmic acid for 1 h, washed with distilled water, and embedded in Epon-812.Samples were cut into ultrathin section (70 nm) using an LKB-V ultramicrotome (Bromma, Sweden) and stained with 0.2% lead citrate and 2% uranyl acetate.The sections were examined with a Hitachi H-600 transmission electron microscope (Hitachi, Tokyo, Japan).

Isolation of total RNA and qPCR
Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions.Real-time quantitative PCR (qPCR) analysis was performed using the Roche LC480 and SYBR RT-PCR kits (DBI Bioscience, Ludwigshafen, Rhineland-Palatinate, Germany) according to the manufacturer's instructions.The data were normalized according to the level of β-actin expression in each sample.

Western blotting
Total proteins were extracted by RIPA buffer (Beyotime Biotechnology, P0013E) containing complete protease inhibitors (Roche Applied Sciences).The protein concentration was quantified using the Pierce ® BCA Protein Assay Kit (Pierce, 23225).Samples containing the same amounts of proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8-15% acrylamide gels and transferred to polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membranes.The membranes were blocked in 5% non-fat milk and incubated with indicated primary antibodies at 4 ℃ overnight.After incubating with horseradish peroxidase-conjugated secondary antibody, the bands were visualized by the ECL detection system (Millipore).

Nuclear extraction
Cells were incubated in serum-free media for 24 h, washed twice with cold PBS, and scraped into 1 ml cold PBS.Cells were harvested by centrifugation (15 s) and incubated in two packed cell volumes of buffer A (10 mM HEPES, pH 8, 0.5% Nonidet P-40, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, and 200 mM sucrose) for 5 min at 4 °C with the flipping of the tube.The crude nuclei were collected by centrifugation (30 s); pellets were rinsed with buffer A, resuspended in one packed cell volume of buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, and 1.0 mM DTT), and incubated on a shaking platform for 30 min at 4 °C.Nuclei were centrifuged (5 min), and supernatants were diluted 1:1 with buffer C (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, and 1 mM DTT).Cocktail protease inhibitor tablets were added to each type of buffer.Nuclear extracts were snap-frozen in liquid nitrogen and stored at − 70˚C until use.

Protein purification
For in vitro pull-down assays, Erbin was cloned into the expression vector pGEX-4T-1 (Amersham Pharmacia, Buckinghamshire, UK) and was expressed in Rosetta (DE3) pLys (Novagen) E. coli cells.The recombinant proteins were purified on glutathione-sepharose bead (Pharmacia, Buckinghamshire, UK) columns to obtain relatively pure GST-Erbin.His-ATG16L, and His-TFEB were induced in the same manner and purified on the Ni-NTA columns (Qiagen, Germany).For the in vitro pull-down assays, GST-Erbin, and His-TFEB were incubated together in various combinations.After a short incubation, the reaction systems were immunoprecipitated using agarose-immobilized indicated antibodies or protein A/G agarose beads and were then analyzed by western blotting.

Histological analysis
The liver and lung tissues were harvested to observe morphologic alterations at 6 h, 24 h, and 48 h after CLP.The lung's right middle lobe and the liver's right lobe were excised, washed, and fixed with 4% (v/v) paraformaldehyde for 24 h at 4 °C.The liver and lung tissues were embedded in paraffin, sectioned at 4 μm thickness, dewaxed and rehydrated, and stained with hematoxylin and eosin (H&E) solution (hematoxylin, MHS16; eosin, HT110132; Sigma-Aldrich, USA) to estimate pathological damages.The stained slides were then observed with a light microscope, and the digital micrographs were taken for analysis.Histologic changes were evaluated by a pathologist blinded to the experiment.The histological score of the lungs was calculated according to our study [29].

Serum analysis
The blood was collected from the abdominal aorta, and the serum was obtained following centrifugation (1000 × g for 10 min).The levels of TNF-α, IL-6, IL-1β, HMGB1, and IL-10 in serum were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).The absorbance was measured at 450 nm using an ELISA reader (BioTek Instruments, Inc., USA).

Statistical analysis
Data were expressed as mean ± SEM.Statistical significance was analyzed with one-way analysis of variance (ANOVA) followed by post hoc (Bonferroni t); P values < 0.05 were considered significant.We used SPSS 23.0 to perform the statistical analysis.

Erbin plays an anti-inflammatory protective role in polymicrobial sepsis animals and MDP-treated BMDMs
To investigate the physiological role of Erbin in the inflammatory response to sepsis and sepsis-induced organ injury, we employed the CLP to establish a polymicrobial sepsis animal model, which has been widely used in previous studies [19].As shown in Fig. 1A, CLP induced the production of many pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-6, IL-1β, and high mobility group box 1 (HMGB1) in the serum of wildtype (WT) mice, which were markedly elevated in Erbin knockout mice.In the same experiments, CLP-induced anti-inflammatory cytokine IL-10 production was much lower in the Erbin knockout CLP mice then in the WT mice.These results suggested that Erbin deficiency aggravated sepsis-induced inflammatory response.Furthermore, H&E staining showed that Erbin-knockout aggravated liver injury, which was indicated by multiple foci of hepatocyte necrosis, inflammatory cell infiltration, and fatty change in the liver of CLP mice (Fig. 1B).Consistently, the secretion of aspartate aminotransferase (AST) and alanine transaminase (ALT), which are biochemical enzymes of hepatocytes, is much increased in the serum of Erbin knockout mice (Fig. 1C).We also investigated the effect of Erbin-knockout on lung histopathology in sepsis mice.H&E staining showed that CLP-induced severe pathological changes, including pulmonary capillary congestion, pulmonary interstitial edema, mass inflammatory cells infiltration into the alveolar space and lung interstitium, as well as alveolar wall thickening in WT mice, which were dramatically enhanced in the lung tissues of CLP-challenged Erbin knockout mice (Fig. 1D).Moreover, we have also employed a scoring system to assess the degree of lung injury [20].As shown in Fig. 1E, the quantitative scoring of histological lung injury in Erbin knockout mice was markedly increased compared with that in WT mice after CLP.Additionally, the BALF protein concentration and wet/dry ratio, two widely used indicators of pulmonary vascular permeability, were significantly increased in CLP-challenged Erbin knockout mice compared to those sham WT mice (Fig. 1F, G).We also detected the ratio of the number of polymorphonuclears (PMNs) relative to the total cells in the BALF.Compared to the WT mice, the PMNs/total cells ratio in Erbin knockout mice was dramatically increased after CLP (Fig. 1H).These results suggested that Erbin-deficiency triggers aggravated liver and lung injuries in CLP-challenged mice.Finally, we examined the rates of CLP-induced septic lethality of both WT and Erbin knockout mice, showing that Erbin deficiency decreased the survival rate of sepsis mice (Fig. 1I).
In vitro, we isolated BMDMs from Erbin fl/fl and Erbin fl/fl/Lyz2−Cre mice and primed the cells with MDP for 6 h.Similar to in vivo results, the pro-inflammatory cytokines TNF-α, IL-6, IL-1β, HMGB1, and the antiinflammatory cytokine IL-10 levels were increased in the MDP-treated wild-type supernatant BMDMs, and Erbin deficiency increased the pro-inflammatory cytokines and decreased the IL-10 production (Fig. 1J).All these results suggested that Erbin deficiency increased sepsis-induced inflammatory response, aggravated organ injuries, and reduced survival rates in CLP-challenged mice, indicating an anti-inflammatory protective role of Erbin in sepsis and MDP-treated BMDMs.

Erbin deficiency exacerbates autophagy impairment in sepsis mice and MDP-treated BMDMs
To reveal the further mechanism of Erbin in sepsis, we investigate the effects of Erbin on autophagy in polymicrobial sepsis mice and MDP-treated BMDMs.The liver and lung tissues were dissected at 6 h, 24 h, and 48 h after CLP.Transmission electron microscopy (TEM) experiments showed that more autophagosomes were observed in the liver or lung of Erbin knockout CLP mice than WT CLP (Fig. 2A-D).Furthermore, we have examined the protein levels of several critical components in the autophagy pathway.The results showed that CLP increased LC3-II and SQSTM1 (p62) protein levels in both liver and lung of WT mice, which further accumulated in Erbin-knockout mice (Fig. 2E, F).Generally, the increased conversion of LC3-I to LC3-II represents the enhancement of autophagy.However, the failure of degradation of LC3-II at the lysosomal stage could also lead to the accumulation of LC3-II, resulting in the illusion that autophagy flow is blocked or autophagy is enhanced.De Wet et al. revealed that the increase of p62 level could be detected in autophagy-deficient tissue cells, indicating that the decrease of p62 level accompanies the enhancement of autophagy, but the inhibition of autophagy increases p62 instead [21].Therefore, we evaluated autophagy status by treating with rapamycin (RAPA, an autophagy agonist) or CQ, a lysosomal inhibitor, and detected LC3-II and p62 protein levels, respectively.CQ treatment increased the level of LC3-II and p62 in liver or lung tissues compared with CLP mice.Compared with WT CLP mice treated with CQ, Erbin knockout caused a further increased LC3-II or p62 in the liver and lung tissues of CLP mice (Fig. 2G, H).Additionally, in vitro experiments, Erbin fl/fl and Erbin fl/ fl/Lyz2−Cre BMDMs were exposed to 10 μg/ml MDP for 6 h.The TEM experiments showed that MDP treatment increased autophagosome numbers in Erbin fl/fl BMDMs, and Erbin-knockout caused further more autophagosome numbers in MDP-treated BMDMs (Fig. 2I, J).Immunofluorescence experiments also showed MDP treatment increased LC3-I/II staining in Erbin fl/fl BMDMs, and the LC3-I/II staining was further increased after Erbin ablation in MDP-induced BMDMs (Fig. 2K).We used RAPA as positive control, results showed that RAPA could induce complete autophagic flux in BMDMs, with the increased levels of LC3II and the decreased levels of p62.However, MDP treatment increased the protein levels of LC3-II as well as p62, and Erbin-knockout caused further accumulation of LC3-II and p62 proteins after MDP treatment, with no significant changes in AGT5, ATG7, Beclin1 protein levels (Fig. 2M).To study autophagic flux, CQ was used to inhibit autophagy, results showed that the levels of LC3-II and p62 markedly increased in MDP + CQ treated Erbin fl/fl/Lyz2−Cre BMDMs than those in MDP + CQ treated Erbin fl/fl BMDMs (Fig. 2N).Those results indicated that Erbin deficiency could result in lysosomal dysfunction, damaged autophagosomes degradation and the accumulation of LC3-II and p62 proteins.

Erbin alleviates sepsis-induced inflammatory response and organ injury by regulating the autophagy-lysosome pathway (ALP)
To determine whether Erbin-mediated autophagy regulates sepsis-induced inflammatory response and organ injury, we investigated the level of inflammatory cytokines TNF-α, IL-6, IL-1β, HMGB1, and IL-10 in polymicrobial sepsis mice and MDP-treated BMDMs with CQ treatment.The CQ treatment caused a further aggravated inflammatory response, as indicated by the increased levels of pro-inflammatory cytokines of TNF-α, IL-6, IL-1β, and HMGB1, and the decreased levels of anti-inflammatory cytokine IL-10.Importantly, Erbin deficiency further increased the inflammatory response via CQ treatment in vitro and vivo compared with Erbin fl/fl BMDMs or WT CLP mice treated with CQ, respectively (Fig. 3A, B).The H&E staining experiments Fig. 2 Erbin deficiency exacerbates autophagy impairment in sepsis mice and MDP-treated BMDMs.A, C WT and Erbin −/− mice were subjected to CLP 6 h, and the mouse liver and lung tissues were examined by transmission electron microscopy (TEM).B, D Autophagosomes were quantified by analysis of (A) and (C) results.The arrow indicates autophagosomes.Scale bar: 1 μm.E Western blotting was used to estimate the protein levels of LC3-I/II and p62 at 6 h, 24 h, and 48 h after CLP in mice liver tissue.F Western blotting was used to estimate the protein levels of LC3-I/II and p62 at 6 h, 24 h, and 48 h after CLP in mice lung tissue.G CQ (60 mg/kg) was used as a pretreatment before CLP to inhibit autophagosome-lysosome fusion.The protein expression of LC3-I/II and p62 in liver tissues were detected by western blotting.H CQ (60 mg/kg) was used as a pretreatment before CLP to inhibit autophagosome-lysosome fusion.The protein expression of LC3-I/II and p62 in lung tissues was detected by western blotting.I BMDMs were examined by transmission electron microscopy (TEM).The arrow indicates autophagosome.Scale bar: 1 μm.J Autophagosomes were quantified by analysis of results of (I).K The LC3-I/II fluorescence was detected by confocal microscopy, scale bar: 50μm.L The LC3-I/II fluorescence staining was quantified by analysis of results of (K).M Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP (6 h), and the western blotting was used to estimate the protein levels of LC3-I/II, p62, Beclin1, ATG5, and ATG7.200 nM RAPA was used as a positive control for autophagy.N CQ (5 μM) was used as a pretreatment before MDP to inhibit autophagosome-lysosome fusion.The LC3-I/II conversion and p62 accumulation were detected by western blotting in BMDMs.*P < 0.05, **P < 0.01, ***P < 0.001 from ANOVA followed by Tukey's post hoc test showed that the inhibition of autophagy by CQ treatment caused severe injury of the liver and lung tissues of WT mice after the CLP challenge, which the injury scores were dramatically aggravated in the liver and lung tissues of CLP-challenged Erbin knockout mice (Fig. 3C, D).Those observations indicated that Erbin promoted the autophagy process and alleviated sepsis-induced inflammatory response and organ injury.

Erbin improves lysosomal biogenesis in MDP-treated BMDMs
To further explore the regulatory mechanism of Erbin in autophagy process during sepsis.We detected the lysosomal acidic microenvironment by using LysoTracker Red stain.Compared with MDP-treated Erbin fl/fl BMDMs, the fluorescence intensity was decreased in MDP-treated Erbin-knockout BMDMs, indicating that Erbin deficiency reduced the acidification of lysosomes (Fig. 4A,   B).Interestingly, we detected the protein levels of vacuolar-type proton pumping ATPase (V-ATPase) subunits ATP6V1A and ATP6V1B2.Compared with MDP-treated Erbin fl/fl BMDMs, the ATP6V1A and ATP6V1B2 levels were reduced in MDP-treated Erbin fl/fl/Lyz2−cre BMDMs.The levels of lysosome proteins lysosome-associated membrane protein-2 (LAMP-2) and CTSB were significantly decreased in MDP-treated Erbin fl/fl/Lyz2−cre BMDMs than that in MDP-treated Erbin fl/fl BMDMs (Fig. 4C).Those results indicated that Erbin deficiency decreased lysosome numbers and lysosomal degradative function.

Erbin promotes TFEB nuclear translocation and transcription activity
TFEB is located in the cytoplasm in the form of phosphorylation, and dephosphorylated TFEB transfers from cytoplasmic into intracellular nucleus, activating the Fig. 3 Erbin-mediated autophagy response alleviated sepsis-induced inflammatory response and organ injury.A Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were pretreated with CQ (5 μM) for 12 h and then treated with MDP for 6 h.The TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured in the supernatant of Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs.B The Erbin −/− and WT littermate mice were pretreated with CQ (60 mg/kg) before CLP 1 h, and mice were euthanized after CLP 6 h.The serum TNF-α, IL-6, IL-1β, HMGB1 and IL-10 levels were measured by ELISA.C, D Histopathologic changes in the liver and lung tissues were detected by H&E staining.Each group had 10 mice.Data shown are representative of 3 independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 from ANOVA followed by Tukey's post hoc test expression of lysosomal and autophagy-related genes.We found that Erbin knockout increased the phosphorylated TFEB/S211 level in MDP-treated Erbin fl/fl/Lyz2−cre BMDMs compared with MDP-treated Erbin fl/fl BMDMs group, and Erbin over-expression inhabited the phosphorylated TFEB/S211 level in THP-1macrophages (Fig. 5A,  B).Consistently, the subcellular fractionation experiments showed that Erbin over-expression increased the cytoplasm-to-nucleus translocation of TFEB, and Erbin knockdown decreased the nuclear translocation of TFEB after MDP stimulation (Fig. 5C-E).These results indicated that Erbin promotes the TFEB nuclear translocation.The mRNA levels of the TFEB target genes VPS18, SYNJ2, and HSP8 were significantly decreased in MDP-treated Erbin-knockdown THP-1 macrophages compared to the control group (Fig. 5F).Those results suggested that Erbin promotes TFEB nuclear translocation and transcription activity.

Discussion
The present study demonstrates that Erbin deficiency disrupts autophagy progress, thus amplifying the inflammatory response and organ injury during sepsis.Importantly, its impaired acidic microenvironment  Erbin deletion [17].Shen et al. found that Erbin-deficient mice exhibited a more severe inflammatory response and intestinal barrier disruption of colitis mice [22].Here, we found that Erbin deficiency aggravated sepsis-induced inflammatory responses, organ dysfunction, and even death.Therefore, Erbin might be an early warning indicator for sepsis, which uncovers the therapeutic potential of Erbin in sepsis.Autophagy serves as an immune response to microbial infection [23], and several previous investigations have suggested autophagy activity is increased in response to sepsis insult based on the increased autophagosomes and the conversion of LC3-I to LC3-II.However, this assumption is not necessarily correct, as the presence of numerous autophagosomes or the increased LC3-II could result from an increase in formation or a decrease in clearance [24].For example, in a pancreatitis model, the autophagosome formation and LC3-I/II were significantly increased after LPS treatment.However, this resulted from the blockade fusion of autophagosomes and lysosomes rather than the induction of autophagy [25].SQSTM1/ p62, a stress-induced cellular protein, acts as a receptor for selective autophagy and is considered to be a good reflection of clearance function, with the LIR domain interacting with multiple sites of LC3, and subsequently degraded along with LC3 and the ubiquitinated protein aggregates [26].Thus, failing to efficiently degrade p62 suggested that autophagy should not be sufficient, possibly due to impaired lysosomal/autolysosomal functions or decreased lysosome numbers.In the present study, we found that Erbin alleviated the dysfunction of ALP caused by sepsis, which may account for the protective role of Erbin in sepsis.
Autophagy dysfunction is considered the potential toxic mechanism of sepsis.Saitoh et al. reported that Atg16L1 deficiency increased caspase-1 activation and IL-1β processing in LPS-stimulated macrophages [27].Similarly, after endotoxin stimulation, IL-1β production in ATG7-deficient or treated with autophagy inhibitor 3-methyladenine (3-MA) macrophages is enhanced.Chung et al. reported that pretreatment of chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers [28].Autophagic inducer RAPA improved the survival rate, histologic scores, lung wet/dry weight ratio, PaO 2 /FiO 2 , MPO activity, the proinflammatory cytokines TNF-α, HMGB1, IL-6, IL-10, and MCP1 production in septic mice, but there were exacerbated above indicators in sepsis mice pretreated with autophagy inhibitor 3-MA [29].Functionally, in our study, expression patterns of inflammatory cytokine and Fig. 7 Erbin rescues sepsis-induced ALP dysfunction by binding to TFEB, alleviating inflammation response and organ injuries.In this model, the NOD2-NF-κB pathway is activated on DAMPs and PARMs stimulation, resulting in the dysfunction of ALP, especially the disrupted lysosomal acidic environment.In this process, Erbin physically interacts with TFEB and regulates TFEB-14-3-3 and TFEB-PPP3BC complexes stability, which regulates the TFEB nuclear translocation, thus regulating TFEB transcription levels and improving lysosomal biogenesis.Thus, impaired Erbin-mediated ALP leads to an aggravated sepsis inflammation response and a deteriorated prognosis.Those suggested that Erbin's targeting of TFEB might rescue the dysfunctional ALP and alleviate the inflammation response and organ injuries following sepsis organ injuries were significantly increased in Erbin deficiency mice after being treated with CQ.Higher expression of TNF-α, IL-6, IL-1β, and HMGB1 accompanied with aggravated organ injuries were observed in Erbin deficiency after CQ treatment.It indicated that Erbin alleviated sepsis-induced inflammatory responses and organ injuries by rescuing autophagic flux impairment.
Since autophagy is a lysosome-dependent degradation process that includes the early stage of autophagy initiation and the late stage of autophagy degradation [30].Fusion of autophagosomes with lysosomes is necessary for complete autophagic flux [31].Lysosome plays a central role in ALP.The destruction of lysosomes comprises two sides named lysosomal membrane permeabilization (LMP) and lysosomal membrane rupture (LMR) [32].Mutation of LAMP-1 and LAMP-2 influenced autophagosomes' combination with endosomal vesicles to lysosomes, and lysosomal proteases cathepsins are required for subsequent substrate degradation [33].Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase) [34].In Erbin deficiency BMDMs, we found lower levels of CTSB and LAMP2, as well as a more serious disrupted lysosome acidic environment, than the MDP-treated WT BMDMs.These results suggested that Erbin deficiency would destroy the lysosome structure and lysosomal biogenesis to influence the autophagic flux.
Additionally, there are other genes that affect multiple steps of this progress.TFEB was recently discovered as a major regulator of the ALP as well as a potential therapeutic target [35].In addition, PPP3CB-mediated dephosphorylation of TFEB, and YWHA/14-3-3 dissociation were required for TFEB nuclear translocation [36].In the present study, total TFEB expression was increased in response to MDP treatment.Additionally, Erbin deficiency resulted in a marked accumulation of TFEB protein in the cytoplasmic subfraction with a corresponding decline in the nuclear subfraction.We also found that Erbin overexpression promoted TFEB translocation from the cytoplasm to the nucleus after MDP treatment in vitro.Furthermore, co-immunoprecipitation showed that Erbin promoted the dissociation of TFEB from the TFEB-14-3-3 complex and enhanced the interaction between TFEB and PPP3CB.The finding suggested that Erbin-mediated TFEB translocation to the nuclear through the regulation of TFEB-14-3-3 and TFEB-PPP3CB complexes stability.It uncovered the potential molecular mechanism of Erbin regulating autophagy after sepsis insult.
In conclusion, our study demonstrated that Erbin promoted the autophagy process and activated lysosomal biogenesis via direct targeting and activation of TFEB.The cooperatively molecular action rescued autophagy dysfunction caused by sepsis insult and alleviated inflammatory response and organ injuries during the sepsis challenge.Our findings thus reveal novel insight into the regulatory mechanisms of Erbin on autophagy and inflammatory response and provide a potential therapeutic target.

Fig. 1
Fig. 1 Erbin deficiency aggravated inflammatory response and organ injuries and deteriorated prognosis of sepsis.A Erbin −/− mice and WT mice were subjected to CLP procedure and euthanasia of mice after CLP 6 h, 24 h, and 48h.The serum TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured.B, C Histopathologic changes in the liver tissue, serum AST, and ALT levels were detected after CLP.D-H Histopathologic changes and the injury score of lung tissue, the wet/dry ratio, the protein concentration, and the PMN/total cells in BALF were measured.I The survival rates of WT and Erbin −/− mice subjected to CLP were observed.J BMDMs were treated with MDP (6 h), and the TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured in the supernatant of Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs.Each group had 10 mice.The data are representative of 3 independent experiments.*P < 0.05, **P < 0.01, ***P < 0.001 from ANOVA followed by Tukey's post hoc test

Fig. 4
Fig. 4 Erbin enhanced lysosome biogenesis in BMDMs.A BMDMs were treated with 10 μg/ml MDP (6 h) and then stained with LysoTracker Red for 15 min.The fluorescence intensity of treated cells was measured by confocal microscopy.Scale bar: 25 μm.B Fluorescence puncta were quantified by analysis of results of (A).C Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP for 6 h, and western blotting was used to estimate the protein levels of LAMP2, CTSB, ATP6V1A, and ATP6V1B2.*P < 0.05, **P < 0.01, ***P < 0.001 from ANOVA followed by Tukey's post hoc test

Fig. 5
Fig.5 Erbin promotes TFEB nuclear translocation and transcription activity.A Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP for 6 h, and western blotting was used to estimate the protein levels of total TFEB and pTFEB (S211).B THP-1 macrophages were transfected with vector control or HA-Erbin for 36 h prior to western blot assays.C BMDMs were treated with 10μg/ml MDP for 6 h and then incubated with TFEB antibody.The fluorescence intensity of treated cells was measured by confocal microscopy.Scale bar: 25 μm.D THP-1 macrophages were transfected with vector control or HA-Erbin for 36 h.Then, cells were treated with or without MDP (10 μg/ml) for 6 h.Cytosolic and nuclear extracts were prepared and subjected to western blot analyses.Lamin A and β-actin were used as the internal control for nuclear and cytosolic fractions, respectively.E Experiments were performed similarly to those in D, except indicated siRNA was used.F THP-1 macrophages were transfected with siRNA control or si-Erbin for 36 h and treated with or without MDP (10 μg/ml) for 6 h before qPCR analyses.The data shown are representative of 3 independent experiments

Fig. 6
Fig. 6 Erbin binds to TFEB and regulates the stability of TFEB-14-3-3 and TFEB-PPP3CB complexes.A 293T cells were transfected with indicated plasmids.48 h post-transfection, Co-IP and immunoblot analysis were performed with indicated antibodies.B, C Experiments were performed similarly to those in A, except indicated plasmids were used.D Schematic diagram of the full-length and truncated constructs of Erbin.The 293T cells were co-transfected with Myc-TFEB and the indicated truncated Erbin constructs for 48 h.Co-immunoprecipitation and immunoblot analyses were performed with the indicated antibodies.E Schematic diagram of the full-length and truncated constructs of TFEB.The 293T cells were co-transfected with HA-Erbin and the indicated truncated TFEB constructs for 48 h.Co-immunoprecipitation and immunoblot analyses were performed with the indicated antibodies Fang et al.Journal of Translational Medicine (2023) 21:916